Culturing apparatus

ABSTRACT

The present invention relates to a culturing system capable of culturing viable cells while reducing opportunities for infection by bacteria and other microorganisms. This culturing system comprises, in a sterile chamber, a storage unit that respectively stores media and body tissue filler, a cell extraction unit that extracts cells to be cultured from collected body fluid, cell culturing units that are connected to the storage unit and the cell extraction unit, and culture extracted cells in media to produce a body tissue filling composed of cultured cells and body tissue filler, and a sealing unit that seals the produced body tissue filling in a vessel.

TECHNICAL FIELD

The present invention relates to a culturing system for culturing cellsfrom body fluid collected from a patient.

BACKGROUND ART

In recent years, it has become possible to repair areas of missing bodytissue by regenerating bone or other body tissue by replenishing thesite of missing body tissue such as a site of missing bone caused by theremoval of bone tumors or trauma with a scaffold or other body tissuefiller material. Although known examples of scaffolds includehydroxyapatite (HAP) and tricalcium phosphate (TCP), a scaffold composedof a porous calcium phosphate material like β-TCP is used based on theapproach of not allowing foreign objects to remain in the body. If β-TCPis allows to contact osteocytes at a site of missing bone, osteoclastsconsume the β-TCP enabling osteoblasts to form new bone. As a result, aprocess referred to as remodeling takes place. Namely, the scaffold usedto replenish the site of missing bone is replaced by autologous boneover time.

On the other hand, in order to enhance the rate of repair of sites ofmissing bone following surgery, instead of using scaffold directly, bonemarrow liquid collected from the patient is applied to the scaffold toreplenish the site of missing bone. In this case, bone marrow liquidcontaining a large number of mesenchymal stem cells is collected fromthe patient, and after immersing the scaffold in this bone marrowliquid, it is applied to the site of missing bone. However, there is theproblem of being unable to enable an adequate amount of mesenchymal stemcells to adhere to the scaffold simply by immersing the scaffold in thebone marrow liquid alone. Consequently, it has been considered tocultured extracted bone marrow liquid and adhere the mesenchymal stemcells that grow to the scaffold so as to adhere an adequate amount ofmesenchymal stem cells to the scaffold.

Various steps including a culturing step, media replacement step,infection examination step are included in the culturing of bone marrowliquid, and all of these steps have conventionally been performedmanually.

However, manual intervention during these steps and during transferbetween steps has the problem of increasing the risk of contamination bybacteria, fungi and other microorganisms.

DISCLOSURE OF THE INVENTION

In consideration of the aforementioned circumstances, the object of thepresent invention is to provide a culturing system capable of supplyinghealthy cultured cells by reducing opportunities for infection bybacteria and other microorganisms.

In order to achieve the aforementioned object, the present inventionprovides a culturing system comprising, in a sterile chamber, a storageunit that respectively stores media and body tissue filler, a cellextraction unit that extracts cells to be cultured from collected bodyfluid, a cell culturing unit that is connected to the storage unit andthe cell extraction unit and cultures extracted cells in media toproduce a body tissue filling composed of cultured cells and body tissuefiller, and a sealing unit that seals the produced body tissue fillingin a vessel.

According to this culturing system,. collected body fluid is sent to acell extraction unit, and cells to be cultured are extracted in saidcell extraction unit. The extracted cells are sent to a cell culturingunit, and after being mixed with media stored in a storage unit, aregrown by culturing for a predetermined amount of time. In addition, abody tissue filling is produced by adhering the grown cells to a bodytissue filler stored in the storage unit. The produced body tissuefilling is output sealed in a vessel by being sent to a sealing unit. Inthis case, since all of these steps are carried out in a sterilechamber, opportunities for infection by bacteria and othermicroorganisms are reduced through the entire culturing period, therebymaking it possible to provide body tissue fillings in a healthy state.

In addition, the present invention provides the aforementioned culturingsystem that further comprises, in the sterile chamber, an examinationunit that is connected to the cell extraction unit and the cellculturing unit, and at least examines body fluid and cells forinfection.

According to this culturing system, examinations for infection can becarried out on body fluid and cells housed in the cell culturing unit.Since examinations for infection are also carried out within the sterilechamber, opportunities for infection by bacteria and othermicroorganisms in the inspection step are also reduced.

In addition, the present invention provides the aforementioned culturingsystem, wherein cells are examined for infection using media discardedfrom the cell culturing unit.

According to this culturing system, as a result of using media discardedfrom the cell culturing unit, cells can be examined for infectionwithout contaminating the cultured cells. Since media is regularly orintermittently replaced in the cell culturing unit and media that is nolonger necessary is discarded each time, by examining for infectionusing this discarded media, infection of cells that had previouslycontacted that media can be examined reliably.

In addition, the present invention provides the aforementioned culturingsystem, wherein the examinations for infection are carried out on a bodytissue filling produced in the cell culturing unit.

According to this type of cell culturing unit, viable body tissuefillings can be provided more reliably by examining for inspectioneither by sampling the final product in the form of the body tissuefilling by cutting away a portion of said filling, or by using a bodytissue filling produced under the same conditions as the final productfor examination.

In addition, the present invention provides the aforementioned culturingsystem further comprising a recording unit that is connected to theexamination unit and records at least information that includesexamination results on a vessel in which the body tissue filling issealed in the sealing unit.

According to this type of culturing system, since information includingthe results of examinations for infection are recorded on the vessel inwhich the body tissue filling is sealed due to the operation of arecording unit, the person receiving the produced body tissue filling isable to determine the results of examination of said body tissue fillingfrom information recorded on the vessel. Thus, the quality of bodytissue fillings sealed in the vessel is guaranteed.

In addition, the present invention provides the aforementioned culturingsystem, wherein the cell culturing unit comprises a primary culturingunit that cultures extracted cells in media, and a secondary culturingunit that cultures the cultured cells in media together with a bodytissue filler.

According to this type of culturing system, grown cultured cells can beobtained in the primary culturing unit, while a body tissue filling inwhich cultured cells have been grown using the body tissue filler as abase material can be obtained in the secondary culturing unit. Thus,cultured cells or body tissue fillings can be obtained as products. Inaddition, since examinations for infection can be carried out in boththe primary and secondary culturing units, viability can be confirmedmore reliably.

In addition, the present invention provides the aforementioned culturingsystem, wherein the sealing unit seals cells cultured in the primaryculturing unit.

According to this type of culturing system, both body tissue fillingsand cells cultured in the primary culturing unit can be provided in aviable state while avoiding infection by bacteria and othermicroorganisms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing showing and input and output of aculturing system as claimed in a first embodiment of the presentinvention.

FIG. 2 is a block diagram showing the general constitution of theculturing system of FIG. 1.

FIG. 3 is a flow chart for explaining each step of the culturing systemof FIG. 2.

FIGS. 4A and 4B are drawings for explaining the operation of a scaffoldsupply device used in the culturing system of FIG. 1.

FIG. 5 is a flow chart for explaining each step of a culturing system asclaimed in a second embodiment of the present invention.

FIG. 6 is a schematic drawing showing an example of another culturingsystem of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

The following provides an explanation of a culturing system as claimedin a first embodiment of the present invention with reference to FIGS. 1through FIGS. 4A and 4B.

As shown in FIG. 1, the culturing system as described in the presentembodiment outputs a bone filling 5 to which cultured cells are adheredand data indicating the results of examinations for infection in eachstep by being provided with bone marrow liquid 2 collected from apatient, scaffold 3 to which cells contained in said bone marrow liquid2 are adhered, and media 4.

More specifically, as shown in FIG. 2, culturing system 1 as describedin the present embodiment is equipped with a storage unit 6, a cellextraction unit 7, cell culturing units 8 a and 8 b, sealing unit 9 andexamination unit 10 all within a sterile chamber 11.

The aforementioned storage unit 6 is composed of a plurality of vesselsthat separately house scaffold 3 and media 4, respectively. As shown inFIG. 3, media 4 is composed of, for example, minimal essential medium(MEM), fetal bovine serum (FBS) and antibiotic, and these are housedeither in a mixed state or in a state in which they are divided amongseparate vessels 12, 13 and 14. In addition, the vessel that storesmedia 4 is housed in a refrigeration device not shown at a temperatureof, for example, 4° C. The antibiotic is, for example, apenicillin-based antibiotic.

Bone marrow liquid 2 is supplied to cell extraction unit 7. Theaforementioned cell extraction unit 7 is, for example, a centrifuge, andis able to extract bone marrow cells having a large specific gravityfrom bone marrow liquid 2 by receiving bone marrow liquid 2 from avessel in which bone marrow liquid 2 is housed followed by centrifugingthat bone marrow liquid 2.

The cell culturing unit for culturing the aforementioned bone marrowcells is composed of primary culturing unit 8 a and secondary culturingunit 8b. As shown in FIG. 3, primary culturing unit 8 a is respectivelyconnected to the aforementioned cell extraction unit 7 and the vesselhousing media 4. This primary culturing unit 8 a is equipped with aculturing vessel 15 for receiving bone marrow cells extracted in cellextraction unit 7 and media stored in avessel, a stirring device (notshown) that stirs the bone marrow cells and media within culturingvessel 15, and a thermo-hygrostat 16 that maintains the stirred andmixed bone marrow liquid 2 and media 4 at a predetermined temperature(e.g.,37±0.5° C.), CO₂ concentration (e.g., 5% by volume) and otherculturing conditions, and is capable of culturing cells at fixedculturing conditions for predetermined amount of time. In addition, avessel 17 that stores a protease such as trypsin is connected to primaryculturing unit 8 a. As a result, cells adhered to the bottom ofculturing vessel 15 can be detached from culturing vessel 15 bysupplying trypsin to culturing vessel 15 during the culturing step inprimary culturing unit 8 a. Moreover, a centrifuge 18 is also connectedto primary culturing unit 8 a, and this enables cells mixed in thetrypsin to be extracted.

In addition to being connected to primary culturing unit 8 a, secondaryculturing unit 8 b is also connected to a vessel (not shown) housingscaffold 3. Similar to the aforementioned primary culturing unit 8 a,this secondary culturing unit 8 b is equipped with a culturing vessel19, stirring device not shown and a thermo-hygrostat 20. A scaffoldsupply device 21 like that shown in FIGS. 4A and 4B, for example, isprovided in the vessel that houses scaffold 3. This scaffold supplydevice 21 is composed of a shutter 23 provided in a vessel 22 thathouses scaffold 3, and a holding device 24 that holds scaffold 3 fartherup than a lowermost scaffold 3 a so as to only permit dropping of thislowermost scaffold 3 a. Holding device 24 is composed of a piston 25 anda holding rod 26.

Namely, holding device 24 is operated as shown in FIG. 4B from a statein which all scaffolds 3 are placed on shutter 23 as a result of closingsaid shutter 23 as shown in FIG. 4A, and by opening shutter 23 in thestate in which the second scaffold 3 b from the bottom is held byholding rod 26, only the lowermost scaffold 3 a can bemade to drop down.Thus, by operating holding device 24 and opening and closing shutter 23in accordance with a requested amount of scaffolds 3 determinedaccording to the amount of cultured bone marrow cells, a desired amountof scaffolds 3 are loaded into culturing vessel 19.

In addition, media replacement devices 27 and 28 for periodicallyreplacing the media in media vessels 15 and 19 are provided in primaryculturing unit 8 a and secondary culturing unit 8 b. Media replacementdevices 27and 28 are equipped with, for example, a vessel rotatingdevice (not shown) that rotates culturing vessels 15 and 19 about thehorizontal axis. Namely, media 4 inside media vessels 15 and 19 can bedischarged from media vessels 15 and 19 by inclining or inverting mediavessels 15 and 19 as a result of operating the vessel rotating deviceand rotating media vessels 15 and 19 about the horizontal axis.

In addition, a waste media vessel 29 that houses discharged media 4′ isprovided in media replacement devices 27 and 28.

Sealing unit 9 is connected to secondary culturing unit 8 b, and iscomposed to output bone fillings produced in said secondary culturingunit 8b sealed within a sealed vessel 30.

Moreover, specimen culturing unit 31 is connected to secondary culturingunit 8 b, and is composed so that specimens for examination are removedfrom the final products in the form of the bone fillings.

In addition, examination unit 10 is a fully automated, real-timepolymerase chain reaction (PCR) device that is connected to the vesselof the aforementioned bone marrow liquid 2, waste media vessel 29 ofprimary culturing unit 8 a, waste media vessel 29 of secondary culturingunit 8 b and specimen extraction unit 31. In this fully-automated,real-time PCR device, primers are used so as to enable detection of alltypes of contaminants including fungi, bacteria, endotoxins, viruses andmycoplasma.

In the culturing system 1 as claimed in the present embodiment, thestorage unit 6, cell extraction unit 7, cell culturing units 8 a and 8b, sealing unit 9 and examination unit 10 are arranged within sterilechamber 11. Similar to a clean room, for example, the inside of sterilechamber 11 is set to a constant positive pressure, and the amount ofdust per unit volume is limited by a particle counter and so forth.

The following provides an explanation of the operation of a culturingsystem 1 as claimed in the present embodiment composed in this manner.

According to culturing system 1 as claimed in the present embodiment,when bone marrow liquid 2 collected from a patient is placed in the bonemarrow liquid vessel, a portion of said bone marrow liquid 2 is sent toexamination unit 10 where it is examined for infection. The remainder ofbone marrow liquid 2 is sent to cell extraction unit 7 where it iscentrifuged in said cell extraction unit 7 and the bone marrow cells tobe cultured are extracted.

The extracted bone marrow cells are grown to a predetermined number ofcells by being sent to primary culturing unit 8a and cultured.

As shown in FIG. 3, in primary culturing unit 8 a, the bone marrow cellsthat have been sent to this primary culturing unit 8 a and media 4composed of MEM, FBS, antibiotic and so forth that is stored in storageunit 6 are mixed within culturing vessel 15. A portion of media 4 isremoved prior to mixing and then examined for infection by being sent toexamination unit 10.

The mixed bone marrow cells and media 4 are cultured for a predeterminedamount of time in a thermo-hygrostat in the state in which predeterminedculturing conditions are maintained. A judgment is made as to the timewhen the media is to be replaced, and when a predetermined mediareplacement time is reached, media 4′ is discharged to waste mediavessel 29. A portion of media 4′ that is discharged to waste mediavessel 29 is examined for infection by being sent to examination unit10. On the other hand, a judgment as to whether the time for completingprimary culturing has been reached is made for the cells remaining inthe culturing vessel, and if that completion time has not been reached,media 4 is mixed in again and primary culturing is continued. Mixing ofmedia 4, culturing and discharge of media 4′ are repeated until the timeprimary culturing is completed.

In the case the time for completion of primary culturing has beenreached, a quality examination is carried out to determine whether ornot the number of cells has reached the predetermined number, and in thecase the number of cells has not increased sufficiently, the primaryculturing step is carried out again. In the case the number of cells hasbeen judged to have increased sufficiently in the quality examination,trypsin is charged into culturing vessel 15 to detach cultured cellsthat have grown on the bottom of culturing vessel 15. Subsequently, thenumber of cultured cells is adjusted by being applied to ahemocytometer, and then sent to secondary culturing unit 8 b.

In secondary culturing unit 8 b, together with the cultured cells thathave been sent to this secondary culturing unit 8 b being charged intoculturing vessel 19, scaffold 3 housed in storage unit 6 is also chargedinto culturing vessel 19 resulting in dissemination of the culturedcells in scaffold 3. Next, similar to the case of primary culturing,media 4 is supplied to culturing vessel 19 from storage unit 6 and mixedtherein. The supplied media 4 differs from the media 4 used in primaryculturing in that, in addition to MEM, FBS and antibiotic, it is alsomixed with differentiation inducing factor such as dexamethasone orβ-glycerophosphate and nutrients such as vitamin C. Secondary culturingthen begins in the state in which scaffold 3 disseminated with culturedcells in this manner is immersed in media 4. In this secondary culturingstep as well, media 4′ is discharged to waste media vessel 29 atpredetermined media replacement intervals after which it is examined forinfection. When the time for completion of secondary culturing isreached, secondary culturing is completed following another qualityexamination.

When secondary culturing is completed, a bone filling is output fromsecondary culturing unit 8 b that has been adequately grown by usingscaffold 3 as a base material. When the output bone filling is passedthrough specimen extraction unit 31, a portion of the bone filling isremoved as a specimen for examination and then sent to examination unit10 where it is examined for infection. In addition, the remainder of thebone filling is sent to sealing unit 9 where it is sealed within sealedvessel 30. The bone filling that has been output from sealing unit 9 andsealed in sealed vessel 30 is one of the final outputs of culturingsystem 1 as claimed in the present embodiment. In addition, the wastemedia 4′ and specimens sent to examination unit 10 in each of the stepsare examined for infection in examination unit 10, and those inspectionresults are also one of the final outputs of culturing system 1 asclaimed in the present embodiment.

In this manner, according to the culturing system 1 as claimed in thepresent embodiment, a sealed bone filling and the results of infectionexaminations performed during culturing can be obtained simultaneouslysimply by charging bone marrow liquid 2 that has been collected from apatient. Thus, in comparison with the process that was performedmanually in the prior art, the opportunities for infection by bacteria,fungi and other microorganisms can be reduced considerably. As a result,together with being able to provide viable bone fillings, the number oftasks that are performed by workers is reduced, thereby making itpossible to easily produce a large quantity of bone fillings.

Furthermore, following culturing in the primary culturing unit, cellsmay also be sent to sealing unit 9 and output in a sealed state (routeshown with the broken line in FIG. 2).

Next, an explanation is provided of a culturing system 40 as claimed ina second embodiment of the present invention with reference to FIG. 5.

Furthermore, in the explanation of the present embodiment, the samereference symbols are used for those locations in the second embodimentthat share the same constitution as the culturing system 1 as claimed inthe previously described first embodiment, and their explanation isomitted.

Culturing system 40 as claimed in the present embodiment differs fromculturing system 1 as claimed in the first embodiment with respect tobeing provided with a recording unit 41 connected to examination unit10. Recording unit 41 is provided with, for example, a database thatcorrelates and stores groups of examination results for each bonefilling and an identification code 42 for identifying those groups, andan identification code printer. An identification code 42 correspondingto a bone filling contained in sealed vessel 30 can be printed on saidsealed vessel 30 in which said bone filling is sealed.

According to culturing system 40 as claimed in the present embodimentcomposed in this manner, a person who has received a sealed vessel 30imprinted with an identification code 42 is able to extract the group ofexamination results corresponding to said identification code 42 simplyby reading said identification code 42 imprinted on sealed vessel 30,thereby offering the effect of being able to easily determine theexamination results at each stage of the culturing process for each bonefilling in a form that corresponds with said bone filling.

Furthermore, the information stored in the database corresponding toidentification code 42 may include not only the results of infectionexaminations for said bone filling, but also name, gender, age, date ofbirth and other identification data for identifying the patient,culturing conditions and culturing times that indicate a culturing log,and the results of examinations other than infection examinations, suchas the results of quality examinations and so forth.

In addition, the transport devices between each of the units ofculturing systems 1 and 40 arranged within sterile chamber 11, althoughnot specifically indicated, should be composed by typical transportdevices such as conveyors and manipulators. In addition, a roller pumpand so forth can be employed for the device that supplies liquid media 4and so forth.

In addition, as examples of components added to media 4, substances thatcontribute to growth such as growth factors such as cytokines,concentrated platelets, BMP, FGF, TGF-β, IGF, PDGF, VEGF, HGF andcompounds thereof may be blended into media 4. In addition, hormonepreparations such as estrogen and nutrients such as vitamins may also beblended into media 4.

In this case, the blending ratios of these growth factors, hormonepreparations or nutrients should be determined corresponding to thedegree of activity of the bone marrow liquid 2 to be cultured. Inaddition, although penicillin-based antibiotics are used for theantibiotic, cephams, macrolides, tetracyclines, fosfomycines,aminoglycosides, new quinolones and other arbitrary antibiotics may beused instead of these penicillin-based antibiotics.

In addition, although the explanation of the aforementioned embodimentsusing the example of bone marrow liquid 2 for the body fluid to becultured, peripheral blood or placental blood may be used in its place.In addition, in this specification, liquids that contains somatic cellssuch as ES cells, somatic stem cells, mesenchymal cells, bone cells andchondrocytes are also referred to as body fluids for the sake ofconvenience. Moreover, the cells may be autologous cells or heterologouscells.

In addition, although bone was used as an example of body tissue in theaforementioned embodiments, fillings of other body tissues may also beproduced. In this case, any arbitrary material having affinity with bodytissue can be used for the body tissue filler, and a material that isabsorbed by the body is even more preferable. In addition, this materialmay also be porous. Porous materials refer to porous ceramics, collagen,polylactic acid or porous metals and so forth having biocompatibility,and there are no restrictions on these materials provided they have alarge number of pores. Examples of porous materials that can typicallybe used include calcium phosphate-based ceramics such as apatite andβ-tricalcium phosphate (β-TCP), collagen or polylactic acid and so. Inaddition, calcium-phosphate-based ceramics may be combined withcollagen, or calcium phosphate-based ceramics may be combined withpolylactic acid. β-TCP, collagen and polylactic acid have thecharacteristic of being biodegradable and absorbed by the body, whileapatite has the characteristic of having superior strength. It goeswithout saying that a person with ordinary skill in the art would beable to suitably select and use an appropriate type of porous materialcorresponding to the transplant site and so forth.

In addition, although human intervention has been completely eliminatedfrom the culturing process in each of the aforementioned embodiments, asan example of an alternative to this, as shown in FIG. 6, bone filling5, cells for examination 43 and media for examination 44 may be outputfrom culturing system 1, while examinations for infection by fungi,bacteria and other microorganisms may be performed by an examinationdevice 45 provided outside culturing system 1.

In addition, the concrete structures of each of the constituent devicessuch as scaffold supply device 21 in each of the aforementionedembodiments represent examples of one manner of composing the presentinvention, and the present invention is not limited to these.

Industrial Applicability

As has been explained above, according to the culturing system asclaimed in the present invention, since body tissue fillings can beproduced simply by charging a body fluid without requiring humanintervention in a sterile chamber, the effect is obtained of being ableto provide viable body tissue fillings while reducing opportunities forinfection.

In addition, since the examination results can be obtained along withthe body tissue fillings, the effect is obtained of being able toenhance the reliability with respect to quality of the supplied bodytissue fillings, thereby allowing a user to use the fillings withgreater peace of mind.

1. A culturing system comprising, in a sterile chamber, a storage unitthat respectively stores media and body tissue filler, a cell extractionunit that extracts cells to be cultured from collected body fluid, acell culturing unit that is connected to the storage unit and the cellextraction unit, and cultures extracted cells in media to produce a bodytissue filling composed of cultured cells and body tissue filler, and asealing unit that seals the produced body tissue filling in a vessel. 2.A culturing system according to claim 1, further comprising, in thesterile chamber, an examination unit that is connected to the cellextraction unit and the cell culturing unit, and at least examines bodyfluid and cells for infection.
 3. A culturing system according to claim2, wherein cells are examined for infection using media discarded fromthe cell culturing unit.
 4. A culturing system according to claim 2,wherein the examinations for infection are also carried out on a bodytissue filling produced in the cell culturing unit.
 5. A culturingsystem according to claim 2, further comprising a recording unit that isconnected to the examination unit and records at least information thatincludes examination results on a vessel in which the body tissuefilling is sealed in the sealing unit.
 6. A culturing system accordingto claim 1, wherein the cell culturing unit comprises a primaryculturing unit that cultures extracted cells in media, and a secondaryculturing unit that cultures the cultured cells in media together with abody tissue filler.
 7. A culturing system according to claim 6, whereinthe sealing unit seals cells cultured in the primary culturing unit.